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Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

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Published on: November 29, 2014

Targeting a Pleckstrin Homology Domain with a Lysine-Reactive Covalent Binder.

Rebekah M West¹, Radu Costin Bizga Nicolescu¹, Paul Brear²

¹Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.

Journal of Medicinal Chemistry

|May 14, 2026

Summary

Researchers developed a new way to inhibit Bruton's Tyrosine Kinase (BTK) by targeting its PH domain. This novel approach avoids the ATP binding site, potentially offering a better therapeutic index for hematological malignancies.

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Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

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Published on: October 26, 2015

Area of Science:

Biochemistry
Medicinal Chemistry
Pharmacology

Background:

Bruton's Tyrosine Kinase (BTK) is a key target for treating hematological malignancies, with existing inhibitors approved by the FDA.
Current BTK inhibitors target the ATP binding site, which is highly conserved across kinases, limiting the therapeutic index.
The PH domain of BTK mediates its membrane recruitment and activation, presenting an alternative inhibition target.

Purpose of the Study:

To explore a novel strategy for BTK inhibition by targeting the PH domain.
To identify and develop inhibitors that bind to the PH domain and disrupt BTK membrane localization.
To assess the potential for a more general approach to PH domain inhibition.

Main Methods:

Fragment-based drug discovery was employed to identify initial binders to the BTK PH domain.
Structure-activity relationship studies and fragment growth were conducted to optimize inhibitors.
Crystallography was used to determine 27 crystal structures, elucidating binding modes.
pKa values of targeted lysines in BTK and other PH domains were evaluated.

Main Results:

A fragment was identified that covalently modifies a lysine within the inositol phosphate (PIP3) binding site of the BTK PH domain.
This fragment was shown to inhibit the binding of a PIP3 headgroup analog to the PH domain.
A best-in-class analog, compound 24, was developed through fragment optimization.
Evaluation of pKa values suggests this covalent modification strategy may be applicable to other PH domains.

Conclusions:

Targeting the PH domain offers a novel and potentially more selective approach to BTK inhibition compared to ATP-competitive inhibitors.
Compound 24 represents a promising lead optimized through structure-based design.
The identified mechanism of covalent modification of a lysine in the PIP3 binding site may be generalizable to inhibit other PH domain-containing proteins.